Heat shock protein 90 (Hsp90) is classically thought as the core component of a chaperone system (herein referred to as the `Hsp90 system') that modulates the activation of multiple signaling proteins in eukaryotic cells. We have discovered a new and unanticipated role for the Hsp90 system in the global regulation of Rab GTPases that control the assembly of tether and fusion complexes that direct membrane vesicular traffic in the exocytic and endocytic pathways, a result now strongly supported by a comprehensive systems analysis of Hsp90 function in yeast. This proposal focuses on understanding the potential, but as yet unknown role(s) of the Hsp90 system in controlling Rab-dependent processes. We now propose the general hypothesis that the Hsp90 system performs a chaperone function related to that observed in classic signaling pathways- to modulate the fold of a number of mature but largely unknown clients populating the Rab-dependent tethering-fusion machineries. We propose the enabling concept of `trafficking homeostasis'- the biological process of maintaining the functionality of components of Rab-regulated oligomeric tethering-fusion complexes that balance protein and lipid cargo flux with the capacity of these trafficking machineries to direct the activity of exocytic and endocytic compartments. Because a role for the Hsp90 system in Rab-dependent tether-fusion events is a central theme in membrane trafficking and therefore in the general membrane biology of the cell, it now becomes imperative to understand the mechanism(s) by which the Hsp90 system regulates these protein interactions- results that will significantly advance the field of cell biology. We propose to characterize the role of the Hsp90 system in regulating the activity of guanine nucleotide dissociation inhibitor (GDI), a general factor controlling recycling of all Rab GTPases (Aim 1), and the tethers p115 and COG involved in vesicle trafficking in the early secretory pathway (Aim 2). Each aim focuses on a Rab- dependent machine that current evidence suggests requires the Hsp90 system for normal function and that leads to loss of trafficking homeostasis when defective in hum disease. PUBLIC HEALTH RELEVANCE: This proposal addresses a new, untapped area in our understanding of the mechanisms that control secretion pathways of mammalian cells. They explore the unanticipated use of `chaperone' systems to control the assembly of complex nanomachines that move protein cargo such as insulin to serum or the cell surface delivery of signaling receptors that control cell proliferation. Given the high impact of these studies on transport of many important proteins traversing the secretory pathway, they will provide critical insight into a broad spectrum of human disorders including type II diabetes, cancer, multiple diseases of hematopoietic lineage, cystic fibrosis, eye disease, congenital disorders of glycosylation (CDGs), mental retardation and Parkinson's disease, among others, as a consequence of inherited dysfunction of these homeostasis pathways.